Directional migration of malignant cells toward a gradient of one or more signaling molecules underlies fundamental steps in metastasis, including local invasion of cancer cells, vascular intravasation, and extravasation of cancer cells at secondary sites. Understanding formation of gradients in complex environments with multiple cells and extracellular matrix molecules remains a central challenge in cell migration not only in cancer but also in normal physiology and other diseases. The challenge of understanding gradient formation and cell migration becomes even more difficult in the disordered cellular and extracellular matrix architecture of a tumor. We will meet this challenge through an integrated systems bioengineering approach combining microscale technologies for cell migration, in vitro and in vivo cellular and molecular imaging, and sophisticated multi-scale computational models. This approach will enable us to investigate gradient formation and cell migration in increasingly complex environments, ranging from a 2D system with defined positions of three different cell types to the disorganized structure of a tumor. Using computational modeling to identify key parameters controlling gradient formation and cell migration, we also will experimentally test and validate interventions to block cell migration, which will provide new targets for anti-metastatic therapies. Our research will focus on gradient formation and cell migration controlled by chemokine CXCL12, a signaling molecule that drives metastasis in more than 20 human cancers. CXCL12 exists as six alternatively-spliced isoforms, four of which are expressed in human breast cancers. We recently have shown CXCL12-isoform specific differences in cell migration, resistance to targeted inhibitors, and correlations with disease recurrence and survival in breast cancer. We propose that CXCL12 molecules bound to the extracellular environment drive cell migration, a process referred to as haptotaxis, and differences in binding to the extracellular matrix underlie isoform-specific differences in gradient formation and cell migration. To investigate CXCL12 isoforms in cell migration, we will complete the following specific aims: 1) derive basic cell migration response parameters under simple, defined gradients; 2) using tissue-like geometries, test effects of extracellular matrix composition on migration potency of CXCL12 isoforms; and 3) Quantify in vivo migration in tumor environments with different CXCL12 isoforms. Collectively, this research will advance knowledge of gradient formation in cell migration and point to new treatment strategies for targeting CXCL12 in cancer.